Particulate methane monooxygenase, pMMO, catalyzes the oxidation of methane to methanol using dioxygen. Recently we showed that one of the EPR-detectable sites is a type 2 site (J. Am. Chem. Soc. 119, 5073-4, 1997). The unusual signal observed by us (g-parallel = 2.28, no hyperfine structure) and others (J. Am. Chem. Soc. 118, 12766-76, 1996) following addition of ferricyanide to membrane fractions has also been observed from type 3 copper of ascorbate oxidase or laccase reacted with ferrocyanide (Sakurai, Inorg. Chim. Acta 195, 255-8, 1992; Biochem. J. 284, 681-5, 1992). Most likely, upon addition of ferricyanide to samples with high pMMO concentrations, copper(I) is oxidized to copper(II) and ferrocyanide is formed. Excess ferrocyanide may also be formed from other reducing equivalents in the membrane fraction or cells. Then the isotropic EPR signal, which is similar to the signal from Cu(2+)Fe(CN)62- appears, followed by the unusual EPR signal with g-paralle l = 2.28 upon incubation of the sample at -20?C. One interpretation of the unusual EPR signal is that interstitial sites are filled by low molecular weight molecules like water to give axial symmetry. The formation of this unusual signal is consistent with, but not proof of, a type 3 or even trinuclear copper site. However, this signal is, most likely, not from an intrinsic trinuclear site as proposed. In this study EPR spectra were obtained for the type 2 copper site in pMMO grown on K15NO3 and 63Cu (NO3)2. Since the nitrogens bound to the type 2 copper(II) are 15N instead of 14N, the superhyperfine pattern changes because I=1/2 for 15N and I=1 for 14N. Also, the spectra are better resolved because A(15N)/A(14N) = 1.4. The five-line pattern for the MI=-2 line in the g-parallel region of the low frequency (3.4 GHz) S-band spectrum is consistent with four approximately equivalent nitrogen atoms bound to cupric ion. At X-band, the MI=-2 region is separated into two very similar lines of almost equal intensity. Both of these lines are consistent with very similar coordination sites for type 2 copper(II) in which cupric ion is bound to four nitrogen donor atoms in both sites. GRANT NUMBER: P41 RR01008-23 (03/01/98 TO 02/28/99) 104